Centromeres are essential to genome inheritance. Abnormal centromere function is associated with birth defects, infertility, and cancer. Human centromeric (CEN) chromatin typically forms on alpha satellite DNA, a 171bp monomeric sequence that is tandemly organized into large multi-megabase arrays. More than half of endogenous human chromosomes have two distinct alpha satellite arrays, either of which can be the site of centromere assembly. Using Homo sapiens chromosome 17 (HSA17) as a model, we demonstrated that in most individuals, the centromere is formed at the primary alpha satellite array. Less frequently (30%), the centromere assembles at the alternative array. We showed that centromere location is dictated by genomic variation within the primary alpha satellite array. When the array contains extensive size and sequence variation, the centromere is usually assembled at the alternative array nearby. However, if the centromere forms at a variant array, the kinetochore is architecturally flawed, resulting in chromosome aneuploidy. The molecular basis for how genomic variation affects centromere assembly and maintenance is not clear. Most human chromosomes must choose between two (or more) locations at which to build a stable centromere, so our work will address a fundamental gap in the knowledge of basic processes governing centromere choice and chromosome stability. In this proposal, we will define molecular links between human centromere assembly and genomic variation in alpha satellite DNA. The proposed work will test the hypothesis that alpha satellite arrays containing variant higher order repeat (HORs) are sub-optimally organized for proper kinetochore assembly. We also postulate that variant HORs produce unstable transcripts that cannot interact appropriately with centromere proteins (CENPs). The experiments in this proposal will define the altered molecular relationship of alpha satellite variation and centromere protein assembly by: 1) mapping alpha satellite long-range organization of HORs and CENPs and characterizing RNA-CENP interactions at normal and defective centromeres, 2) distinguishing if variant arrays are unable to recruit versus retain CENPs, 3) using human artificial chromosome assembly assays to test the ability of variant arrays to assemble centromeres de novo, and 4) repairing defective centromeres using CRISPR engineering. This proposal will address mechanisms of centromere choice and assembly by focusing on the largely unexplored area of genomic variation within highly repetitive DNA.